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Molecular Analysis of Phenanthrene Degradation By MOLECULAR ANALYSIS OF PHENANTHRENE DEGRADATION BY COMAMONAS TESTOSTERONI GZ38 AND ACIDOVORAX SP. GZ39 By KAREN PESCE A dissertation submitted to the Graduate School-New Brunswick Rutgers, The State University of New Jersey in partial fulfillment of the requirements for the Degree of Doctor of Philosophy Graduate Program in Environmental Science written under the direction of Professor Gerben J. Zylstra and approved by _________________________ _________________________ _________________________ _________________________ _________________________ New Brunswick, New Jersey October, 2008 ABSTRACT OF THE DISSERTATION Molecular Analysis of Phenanthrene Degradation by Comamonas testosteroni GZ38 and Acidovorax sp. GZ39 by KAREN PESCE Dissertation Director: Professor Gerben J. Zylstra Polycyclic aromatic hydrocarbons (PAHs) are toxic pollutants that are present in a variety of environments. Some bacterial species have the ability to metabolize these compounds and use them as energy sources for growth. Comamonas testosteroni GZ38 and Acidovorax sp. GZ39 were isolated from the Passaic River, NJ, based on their ability to grow on phenanthrene. These isolates are unusual in that they can grow on phenanthrene but not on naphthalene. The genes that code for phenanthrene degradation in GZ38 and GZ39 were identified. The DNA sequence of the genes is unique with respect to other sequences in the GenBank database with a single match to a putative phenanthrene degradation gene cluster from the phenanthrene degrader Alcaligenes faecalis AFK2 (GenBank accession number AB024945). BLAST matches to the putative genes in both operons suggest that phenanthrene degradation proceeds through phthalate. The putative phthalate degradation gene clusters from GZ38 and GZ39 have also been identified and sequenced. RT-PCR analysis of the phenanthrene degradation genes of GZ39 showed that the genes are expressed as one continuous transcript beginning with a ii transposase that is upstream from the start of the phenanthrene operon. The phenanthrene degradation genes of GZ38 are also expressed as one continuous transcript but it does not include a transposase located upstream from the operon. For both GZ38 and GZ39, expression of the genes was observed with both phenanthrene and succinate as growth substrates. RT-qPCR results for GZ38 showed that the phenanthrene degradation genes were equally expressed on phenanthrene and succinate. A lacZ assay demonstrated that the promoter region upstream of the phnAb gene in GZ39 is constitutively active. A 5’ RACE assay was performed in order to map the transcription start site of the phenanthrene operon in GZ39. Inactivation of the phthalate dioxygenase in GZ39 resulted in the accumulation of phthalate when grown on phenanthrene and provides strong evidence that phenanthrene degradation proceeds through phthalate. Although the phenanthrene degradation genes of GZ38, GZ39, and AFK2 are nearly identical, this is the first report of functional information relating to these genes. iii ACKNOWLEDGMENTS I would like to thank my advisor, Dr. Gerben J. Zylstra for changing the way I think about science. His excellent advice has helped to bring this project to completion. I would also like to thank the members of my thesis committee, Drs. Elisabetta Bini, Donna Fennell, Lee Kerkhof, and Peter Strom for their helpful advice. I would also like to thank my colleagues from whom I have also learned a lot. In particular I would like to recognize Sinéad Ní Chadhain who was integral to all aspects of my research process as well as to the writing of this document. Sinéad, thank you for being patient and for teaching me how to be a scientist. Hey Dad, now you’re not the only PhD in the family. iv TABLE OF CONTENTS ABSTRACT OF THE DISSERTATION ii ACKNOWLEGEMENTS iv TABLE OF CONTENTS v LIST OF TABLES ix LIST OF FIGURES x Introduction 1. Polyaromatic compounds 1 2. Overview of biodegradative pathways for polyaromatic hydrocarbons 4 2.1 Aerobic bacterial naphthalene degradation 6 2.2 Aerobic bacterial phenanthrene degradation 7 3. PAH catabolic genes of Gram negative bacteria 3.1 nah genes 8 3.2 nag genes 10 3.3 phn genes of Burkholderia sp. RP007 10 3.4 phn genes of Alcaligenes faecalis AFK2 11 4. Phenanthrene degradation by Gram negative isolates 4.1 Burkholderia sp. TNFYE 12 4.2 Sinorhizobium sp. C4 12 4.3 Burkholderia sp. C3 13 5. PAH catabolic genes of Gram positive bacteria 5.1 phn genes of Nocardioides sp. KP7 13 v 5.2 nar genes of Rhodococcus sp. strain NCIMB12038 14 6. PAH degradation by Gram positive bacteria 6.1 Bacillus sp. 15 6.2 Mycobacterium sp. BG1 15 7. Bacterial isolates that degrade multiple PAHs 7.1 Mycobacterium vanbaalenii PYR-1 16 7.2 Sphingomonads 16 8. Overview of GZ38 and GZ39 17 Material and Methods 25 1. Growth of GZ38 and GZ39 25 2. Identification of phenanthrene degradation genes in GZ38 and GZ39 26 3. Nucleotide sequence determination 27 4. Nucleotide sequence upstream of the phenanthrene degradation operon from GZ38 28 5. PCR of phthalate degradation genes from GZ38 29 6. Confirmation of phthalate degradation genes in GZ39 30 7. Identification of phthalate degradation genes in GZ39 30 8. Reverse Transcription PCR 31 9. Reverse Transcription qPCR 32 10. Phenanthrene dioxygenase expression 33 11. Generation of mutants for promoter analysis of the phenanthrene degradation operon in GZ39 35 12. Measurement of promoter activity 36 vi 13. Identification of the transcription start site of the phenanthrene degradation genes in GZ39 37 14. Measurement of salicylate accumulation by GZ39 38 15. Generation of a phthalate dioxygenase knockout mutant for GZ39 39 16. Measurement of phthalate accumulation by the GZ39 phthalate dioxygenase mutant 40 Results and Discussion 42 Chapter 1. Identification and sequence analysis of phenanthrene degradation genes in GZ38 and GZ39 42 1. Nucleotide sequence of phenanthrene degradation genes from GZ38 and GZ39 43 2. Nuceotide sequence upstream of the phenanthrene degradation operon of GZ38 50 3. Discussion 51 Chapter 2. Identification and sequence of phthalate degradation genes in GZ38 and GZ39 67 1. Nucleotide sequence of the phthalate degradation genes of GZ38 67 2. Nucleotide sequence of the phthalate degradation genes of GZ39 71 3. Discussion 73 Chapter 3. Functional analysis of the phenanthrene and phthalate degradation genes in GZ38 and GZ39 91 1. Expression of the phenanthrene dioxygenase in GZ39 91 2. Accumulation of salicylate by GZ39 92 vii 3. Operonic structure of the phenanthrene degradation genes of GZ38 93 4. Operonic structure of the phthalate degradation genes of GZ38 94 5. RT-qPCR of the phenanthrene degradation genes of GZ38 95 6. Operonic structure of the phenanthrene degradation genes of GZ39 97 7. Operonic structure of the phthalate genes of G39 97 8. Promoter activity for the phenanthrene degradation genes of GZ39 98 9. Mapping the transcription start site for the phenanthrene genes in GZ39 100 10. Evidence for the involvement of phthalate metabolism in GZ39 101 11. Discussion 102 Conclusions and Future Directions 123 References 133 Curriculum vita 139 viii LIST OF TABLES Table # Title Page # 1. Best BLASTP matches in the GenBank database to the GZ38 phenanthrene degradation sequence 59 2. Best BLASTP matches in the GenBank database to the GZ39 phenanthrene degradation sequence 60 3. Best BLASTP matches in the GenBank database to the GZ38 phthalate degradation sequence 80 4. Best BLASTP matches in the GenBank database to the GZ39 phthalate degradation genes sequence 81 5. PAH biotransformation by E. coli DH5α cells expressing the phenanthrene dioxygenase from GZ39 107 6. RT-qPCR results for GZ38 calculated as average Ct values for GZ38 cells grown on phenanthrene and succinate 108 7. Calculated Miller Units for the LacZ assay performed on GZ39 109 ix LIST OF FIGURES Figure # Title Page # 1. Structures of selected polyaromatic hydrocarbons 19 2. An example of P450 catalysis of PAHs 20 3. Anaerobic PAH degradation of naphthalene 21 4. Naphthalene degradation pathway 22 5. Phenanthrene degradation pathway 23 6. Gene organization of upper pathway nah genes of Pseudomonas isolates 24 7. Construction of a phthalate dioxygenase mutant in GZ39 41 8. Gene organization of the phenanthrene degradation operon of GZ38, GZ39, and AFK2 61 9. Phylogenetic tree of the phenanthrene dioxygenase large subunit and related sequences 62 10. Phylogenetic tree of the phenanthrene dioxygenase small subunit and related sequences 63 11. Phylogenetic tree of the phenanthrene dioxygenase ferredoxin and related sequences 64 12. Phylogenetic tree of the phenanthrene dioxygenase ferredoxin reductase and related sequences 65 13. Phenanthrene degradation operons of organisms with matches to GZ38 and GZ39 66 x 14. Pathway of phthalate degradation in gram negative bacteria 82 15. Gene organization of the phthalate degradation operon of GZ38 83 16. Gene organization of the phthalate degradation operon of GZ39 84 17. Phylogenetic tree of the phthalate dehydrogenase and related sequences 85 18. Phylogenetic tree of the phthalate decarboxylase and related sequences 86 19. Phylogenetic tree of the phthalate reductase and related sequences 87 20. Phylogenetic tree of the phthalate dioxygenase and related sequences 88 21. Gene organization of the phthalate degradation genes of GZ38 and related operons 89 22. Gene organization of the phthalate degradation genes of GZ39 and related operons 90 23. GC-MS trace of the product of naphthalene biotransformation by E. coli DH5α cells containing the phenanthrene dioxygenase from GZ39 110 24. GC-MS trace of the product of phenanthrene biotransformation by E. coli DH5α cells containing the phenanthrene dioxygenase from GZ39 111 25. RT-PCR analysis of the GZ38 phenanthrene degradation operon 112 26. RT-PCR analysis of the phenanthrene dioxygenase from GZ38 cells that were grown on succinate 113 27. RT-PCR analysis of the GZ38 phthalate operon 114 28. RT-qPCR standard curves 115 29. RT-PCR analysis of the GZ39 phenanthrene degradation operon 116 xi 30. RT-PCR analysis of the phenanthrene dioxygenase from GZ39 cells that were grown on succinate 117 31.
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